In this study, typha tassel (TT) and pussy willow (PW) as biomass sources were used to synthesize carbonaceous materials for enzymatic electrode modification. Carbonization was performed at high temperatures of up to 1000 °C under an inert atmosphere and the resulting carbonized TT (CTT) and PW (CPW) were dispersed in dimethylformamide. CTT and CPW were then characterized by SEM/EDX, MAPPING, FTIR, and XRD to confirm the carbonaceous structures. Four different immobilization strategies were used to demonstrate the use of CTT and CPW together with a glucose oxidizing enzyme (glucose oxidase, GOx), an electron transfer mediator (ferrocene, Fc), and a protective polymer coating (Chitosan, Chit). The effect of the Chit layer was first investigated on the performance of the prepared enzymatic electrodes and it was shown that Chit could help to preserve the GOx activity. The effect of the electron transfer mediator whether in a solution or co-immobilized with GOx was also investigated using a mixture of Fc and CTT or CPW and Fc-only redox active film approaches. The results indicate that when Fc co-immobilized with GOx, a better performance was achieved. The prepared electrodes showed promising results for glucose biosensing with a limit of detection and limit of quantification values of 0.97 mM and 3.2 mM, respectively, operating up to 10 mM glucose. This study presents a comprehensive investigation of different immobilization strategies of GOx on carbonaceous electrodes and provides insight into the possible use of such materials as biomass to bioelectronics approaches.